Neutrophil extracellular traps in bacterial infections and evasion strategies.

Neutrophils are innate immune cells that have a vital role in host defense systems. Neutrophil extracellular traps (NETs) are one of neutrophils' defense mechanisms against pathogens. NETs comprise an ejected lattice of chromatin associated with histones, granular proteins, and cytosolic proteins. They are thought to be an efficient strategy to capture and/or kill bacteria and received intensive research interest in the recent years. However, soon after NETs were identified, it was observed that certain bacteria were able to evade NET entrapment through many different mechanisms. Here, we outline the recent progress of NETs in bacterial infections and the strategies employed by bacteria to evade or withstand NETs. Identifying the molecules and mechanisms that modulate NET release will improve our understanding of the functions of NETs in infections and provide new avenues for the prevention and treatment of bacterial diseases.

The Dual Roles of Activating Transcription Factor 3 (ATF3) in Inflammation, Apoptosis, Ferroptosis, and Pathogen Infection Responses.

Transcription factors are pivotal regulators in the cellular life process. Activating transcription factor 3 (ATF3), a member of the ATF/CREB (cAMP response element-binding protein) family, plays a crucial role as cells respond to various stresses and damage. As a transcription factor, ATF3 significantly influences signal transduction regulation, orchestrating a variety of signaling pathways, including apoptosis, ferroptosis, and cellular differentiation. In addition, ATF3 serves as an essential link between inflammation, oxidative stress, and immune responses. This review summarizes the recent advances in research on ATF3 activation and its role in regulating inflammatory responses, cell apoptosis, and ferroptosis while exploring the dual functions of ATF3 in these processes. Additionally, this article discusses the role of ATF3 in diseases related to pathogenic microbial infections. Our review may be helpful to better understand the role of ATF3 in cellular responses and disease progression, thus promoting advancements in clinical treatments for inflammation and oxidative stress-related diseases.

Comparative untargeted and targeted metabonomics reveal discriminations in metabolite profiles between Mycoplasma capricolum subsp. capripneumoniae and Mycoplasma capricolum subsp. capricolum.

Background: Mycoplasmas are among the smallest prokaryotic microbes that can grow and proliferate on non-living media. They have reduced genomes, which may be associated with a concomitant reduction in their metabolic capacity. Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma capricolum subsp. capricolum (Mcc), both belong to the Mycoplasma mycoides cluster, are significant important pathogenic Mycoplasma species in veterinary research field. They share high degree of genome homology but Mcc grows markedly faster and has higher growth titer than Mccp. Methods: This study investigated the metabolites of these two pathogenic bacteria from the middle and late stages of the logarithmic growth phase through liquid chromatography-mass spectrometry-based metabolomics and targeted energy metabolomics. The multivariate analysis was conducted to identify significant differences between the two important Mycoplasma species. Results: A total of 173 metabolites were identified. Of them, 33 and 34 metabolites involved in purine and pyrimidine, pyruvate metabolism, and amino acid synthesis were found to significantly differ in the middle and late stages, respectively. The abundance of fructose 1,6-bisphosphate, ADP, and pyruvate was higher in Mcc than in Mccp during the whole logarithmic period. Lactate was upregulated in slow-growing Mccp. The pH buffering agent N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] added to media effectively prevented pH reduction and increase bacterial viability and protein biomass. The multivariate analysis revealed that the two Mycoplasma species significantly differed in glucose metabolism, growth factor transport and metabolism, cholesterol utilization, and environmental regulation. Conclusion: The study data are beneficial for understanding the metabolomic characteristics of these two crucial Mycoplasma species and shedding more light on mycoplasma metabolism, and serve as a resource for the pathogenesis and development of related vaccines.

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2.

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.

Complete Genome Sequences of Mycoplasma ovipneumoniae Strains 150 and 274, Isolated from Different Regions in Bosnia and Herzegovina.

Mycoplasma ovipneumoniae is an important pathogen in sheep, goats, and wild ruminants. We sequenced M. ovipneumoniae strains 150 and 274 from Bosnia and Herzegovina. Strain 150 has a circular genome of 1,053,380 bp with 29.15% GC content while strain 274 has 1,081,404 bp with 28.82% GC content.

Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

Identification of a new amino acid mutation in the HN protein of NDV involved in pathogenicity.

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are viral entry proteins and are recognized as the major virulence determinants. Previously, a lentogenic NDV virus (CE16) was derived from a mesogenic strain (CI10) through sequential passages in chick embryos. Whole-genome sequence analysis revealed that the two homologous strains shared the same F protein but differed in HN with two amino acid (aa) substitutions (A215G and T430A). To elucidate the molecular reasons for virulence attenuation, two original plasmids (HN-CI10 and HN-CE16) and two single-point mutants (G215A and A430T) reverse-mutated from HN-CE16 were constructed to analyse the known biological functions of HN. The results showed that the A430T substitution significantly weakened the haemadsorption (HAd) activity, increased the neuraminidase (NA) activity, improved the fusion-promoting activity, and enhanced the cleavage-promoting activity of HN-CE16. However, G215A failed to induce obvious functional changes. Therefore, the aa residue HN430 may play a key role in determining virulence. To test this hypothesis, further studies on A430T were conducted through reverse genetics using an infectious cDNA clone. At the viral level, the A430T-mutated virus showed dramatic promotion of viral plaque formation, propagation, and pathogenicity in vitro and in vivo. This study demonstrates a new virulence site associated with HN protein functions, viral propagation, and pathogenicity. All these findings could lay a foundation for illuminating the molecular mechanism of NDV virulence.

Immune Evasion of Mycoplasma bovis.

Mycoplasma bovis (M. bovis) causes various chronic inflammatory diseases, including mastitis and bronchopneumonia, in dairy and feed cattle. It has been found to suppress the host immune response during infection, leading to the development of chronic conditions. Both in vitro and in vivo studies have confirmed that M. bovis can induce proinflammatory cytokines and chemokines in the host. This consists of an inflammatory response in the host that causes pathological immune damage, which is essential for the pathogenic mechanism of M. bovis. Additionally, M. bovis can escape host immune system elimination and, thus, cause chronic infection. This is accomplished by preventing phagocytosis and inhibiting key responses, including the neutrophil respiratory burst and the development of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) that lead to the creation of an extracellular bactericidal network, in addition to inhibiting monocyte and alveolar macrophage apoptosis and inducing monocytes to produce anti-inflammatory factors, thus inducing the apoptosis of peripheral blood mononuclear cells (PBMCs), inhibiting their proliferative response and resulting in their invasion. Together, these conditions lead to long-term M. bovis infection. In terms of the pathogenic mechanism, M. bovis may invade specific T-cell subsets and induce host generation of exhausted T-cells, which helps it to escape immune clearance. Moreover, the M. bovis antigen exhibits high-frequency variation in size and expression period, which allows it to avoid activation of the host humoral immune response. This review includes some recent advances in studying the immune response to M. bovis. These may help to further understand the host immune response against M. bovis and to develop potential therapeutic approaches to control M. bovis infection.

Genome-Wide Analysis of Mycoplasma dispar Provides Insights into Putative Virulence Factors and Phylogenetic Relationships.

Mycoplasma dispar is an important pathogen involved in bovine respiratory disease, which causes huge economic losses worldwide. Our knowledge regarding the genomics, pathogenic mechanisms, and genetics of M. dispar is rather limited. In this study, the complete genome of M. dispar GS01 strain was sequenced using PacBio SMRT technology and first genome-wide analyzed. M. dispar GS01 has a single circular chromosome of 1,065,810 bp encoding 825 predicted proteins. Twenty-three potential virulence genes and two pathogenicity islands were identified in M. dispar This pathogen was cytopathogenic, could form prolific biofilms, and could produce a large amount of H2O2 Methylation analysis revealed adenine and cytosine methylation across the genome and 13 distinct nucleotide motifs. Comparative analysis showed a high collinearity relationship between M. dispar GS01 and type strain ATCC 27140. Phylogenetic analysis demonstrated that M. dispar is genetically close to M. flocculare and M. hyopneumoniae The data presented in this study will aid further study on the pathogenic mechanisms and evolution of M. dispar.

Genome-Wide Analysis of Mycoplasma bovirhinis GS01 Reveals Potential Virulence Factors and Phylogenetic Relationships.

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis.

Differential Immunoreactivity to Bovine Convalescent Serum Between Mycoplasma bovis Biofilms and Planktonic Cells Revealed by Comparative Immunoproteomic Analysis.

Mycoplasma bovis is a major bovine pathogen that causes considerable economic losses in the cattle industry worldwide. Moreover, M. bovis biofilm can persist in the environment and its host. To date, M. bovis biofilm antigens recognized by bovine convalescent sera and their comparison with planktonic cells have not yet been explored. This study utilized an immunoproteomic approach using two-dimensional electrophoresis, immunoblotting using convalescent bovine serum, and subsequent matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) to identify the immunoreactive proteins expressed in biofilm- and planktonic-grown M. bovis strain 08M. Results showed that M. bovis biofilms and planktonic cells demonstrate differential immunoreactivity to bovine convalescent serum for the first time. A total of 10 and 8 immunoreactive proteins were identified for biofilms and planktonic cells, respectively. To our knowledge, a total of 12 out of 15 had not been reported as immunoreactive proteins in M. bovis, and six were specific to M. bovis biofilms. Three proteins, namely, endoglucanase, thiol peroxidase, and one putative membrane protein, that is, mycoplasma immunogenic lipase A, were identified in planktonic cells and biofilms. Most of the identified proteins were cytoplasmic proteins that were mainly involved in transport and metabolism. Moreover, ATP binding, oxidoreductase activity, and GTP binding were their most representative molecular functions. DnaK and Tuf appeared to be the most interactive immunoreactive agent among the identified proteins. Furthermore, six proteins had potential as serodiagnostic antigens. These data will be helpful to improve our current understanding on the host response to M. bovis biofilms and planktonic cells, which may facilitate the development of novel molecular candidates of improved diagnostics and vaccines to prevent M. bovis infections.

Genome-Wide Analysis of the First Sequenced Mycoplasma capricolum subsp. capripneumoniae Strain M1601.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is a common pathogen of goats that causes contagious caprine pleuropneumonia. We closed the gap and corrected rRNA operons in the draft genome of Mccp M1601: a strain isolated from an infected goat in a farm in Gansu, China. The genome size of M1601 is 1,016,707 bp with a GC content of 23.67%. We identified 915 genes (occupying 90.27% of the genome), of which 713 are protein-coding genes (excluding 163 pseudogenes). No genomic islands and complete insertion sequences were found in the genome. Putative determinants associated with the organism's virulence were analyzed, and 26 genes (including one adhesion protein gene, two capsule synthesis gene clusters, two lipoproteins, hemolysin A, ClpB, and proteins involved in pyruvate metabolism and cation transport) were potential virulence factors. In addition, two transporter systems (ATP-binding cassette [ABC] transporters and phosphotransferase) and two secretion systems (Sec and signal recognition particle [SRP] pathways) were observed in the Mccp genome. Genome synteny analysis reveals a good collinear relationship between M1601 and Mccp type strain F38. Phylogenetic analysis based on 11 single-copy core genes of 31 Mycoplasma strains revealed good collinearity between M1601 and Mycoplasma capricolum subsp. capricolum (Mcc) and close relationship among Mycoplasma mycoides cluster strains. Our genome-wide analysis of Mccp M1601 provides helpful information on the pathogenic mechanisms and genetics of Mccp.

Complete Genome Sequence of Mycoplasma capricolum subsp. capripneumoniae Strain zly1309F, Isolated from Endangered Tibetan Antelope.

Mycoplasma capricolum subsp. capripneumoniae is an important pathogen of goats that causes contagious caprine pleuropneumonia. Here, we report the complete genome sequence of M. capricolum subsp. capripneumoniae strain zly1309F, isolated from a Tibetan antelope (Pantholops hodgsonii) in China.

Complete Genome Sequence of Mycoplasma bovis Strain 08M.

Mycoplasma bovis is a major bacterial pathogen that can cause respiratory disease, mastitis, and arthritis in cattle. We report here the complete and annotated genome sequence of M. bovis strain 08M, isolated from a calf lung with pneumonia in China.

Re-evaluation the immune efficacy of Newcastle disease virus vaccine in commercial laying chickens.

Newcastle disease virus (NDV) infection causes serious problems in laying chickens, like reducing egg production, increasing rate of abnormal eggs in spite of strict vaccination in layer farms program. A new evaluation system is needed to show complete protection of the immunization in laying chickens based on the egg-laying performance, rather than clinical signs of the disease. In this study, laying chickens with different anti-NDV HI (hemagglutination-inhibition) antibody titer after vaccination were divided into different groups. These chickens were then challenged with field isolated highly virulent NDV strains. Results showed that the chickens in low HI titers group (5log2 to 8log2) and medium HI titers group (9log2 to 11log2) had atypical symptoms, produced abnormal eggs, and shed virus. Whereas, with HI titers≥12log2, the chickens were completely protected, and did not show symptoms, or produce abnormal eggs or shed virus. Morbidity, positive viral shedding rate and abnormal egg-rate decreased with increase in pre-challenge HI antibody titer. Our result suggested that 12log2 is the threshold of the HI antibody in providing complete protection to laying chickens under field condition, and protective efficacy is correlated with HI antibody titer. This study provides a valuable reference for the vaccination and control of ND in poultry.

Genetic variation in V gene of class II Newcastle disease virus.

The genetic variation and molecular evolution of the V gene of the class II Newcastle disease virus (NDV) isolates with genotypes I-XVIII were determined using bioinformatics. Results indicated that low homology existed in different genotype viruses, whereas high homology often for the same genotypes, exception may be existed within genotypes I, V, VI, and XII. Sequence analysis showed that the genetic variation of V protein was consistent with virus genotype, and specific signatures on the V protein for nine genotypes were identified. Phylogenetic analysis demonstrated that the phylogenetic trees were highly consistent between the V and F genes, with slight discrepancies in the sub-genotypes. Evolutionary rate analyses based on V and F genes revealed the evolution rates varied in genotypes. These data indicate that the genetic variation of V protein is genotype-related and will help in elucidating the molecular evolution of NDV.

Enhanced production of porcine circovirus type 2 (PCV2) virus-like particles in Sf9 cells by translational enhancers.

OBJECTIVE: To investigate the effect of three translational enhancers for enhancing transgene expression in baculovirus expression vector system using GFP as a reporter gene and selected translational enhancers to increase porcine circovirus type 2 (PCV2) VLPs production. RESULTS: P10UTR (the 3'-untranslated region from the baculovirus p10 gene), Syn21 (a synthetic AT-rich 21-bp sequence) and P10UTR/Syn21 increased the GFP yield by 1.4-, 4- and 4.8-fold, respectively. While IVS (intron from Drosophila myosin heavy chain gene) decreased the GFP yield by 65%. Moreover, the synergy of P10UTR/Syn21 increased the yield of PCV2 VLPs by 4.1 fold (45 µg/10(6) cells) compared with standard baculovirus vector. CONCLUSION: The synergy of P10UTR/Syn21 is a potential strategy to improve the recombinant vaccine production besides PCV2 VLPs in BEVS.

Genomic characterisation of two virulent Newcastle disease viruses isolated from crested ibis (Nipponia nippon) in China.

This paper describes the complete genomic sequences of two virulent Newcastle disease virus (NDV) isolates, Shaanxi06 (prevalent genotype VIId) and Shaanxi10 (novel sub-genotype VIi), from sick crested ibises. The genomes of both isolates were 15,192 nt long and consisted of six genes in the order of 3'-NP-P-M-F-HN-L-5'. The genomes of the two isolates were highly similar to other reference NDV strains. However, some unique features were found in the HN protein of Shaanxi06 and the F gene end of Shaanxi10. Shaanxi06 and Shaanxi10 shared the same virulent motif (112-)R-R-Q-K-R-F(-117) at the F protein cleavage site, which coincided with previous pathogenicity test results. Phylogenetic analysis revealed that both isolates were clustered within class II NDV, with Shaanxi06 in genotype VII and Shaanxi10 in genotype VI. Both isolates shared high homology with the prevalent genotype NDV strains that circulate in fowls and waterfowls. This study is the first to provide genomic information about a novel sub-genotype VIi NDV strain and another genotype VIId virus, which will be useful for subsequent investigations.

Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus.

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.